PURIFYING HOMOGENEOUS SPERMATID SUB-POPULATIONS

纯化同质精子细胞亚群

• 批准号: 6163562
• 负责人: EDWARD E SCHMIDT
• 金额: $ 6.83万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6163562
• 关键词: cell population study; cell sorting; genetic translation; genetically modified animals; green fluorescent proteins; laboratory mouse; messenger RNA; polymerase chain reaction; sperm; sperm analysis; spermatogenesis; technology /technique development

DESCRIPTION (Adapted from applicant's description): Spermiogenesis is a sequential assembly process based on carefully timed translation of stored mRNAs in transcriptionally silent cells; however, the molecular mechanisms that determine when individual mRNAs will be translated are not well understood. A large part of the problem results from our having no reliable method of purifying sub-populations of spermatids at different stages of maturation for molecular analysis. The proposed study aims to resolve this deficiency. The applicant proposes to develop a method that uses transgenic mice that expresses the jellyfish green fluorescent protein (GFP) in post- meiotic male germ cells, in combination with fluorescence-activated cell sorting (FACS), to purify homogeneous sub-populations of staged spermatids. The goals of this project are: 1) Isolate at least 20 spermatid sub- populations by FACS. 2) Evaluate the purity and stage of spermatids represented in each sub-population based on morphological, ultrastructural, and molecular criteria; select 10 homogeneous sub-populations representing different stages spanning all of spermiogenesis. 3) Purify mRNP particle- associated mRNA (stored information) and polysome-associated mRNA (active information) from each of these homogeneous sub-populations, prepare a cDNA library from each mRNA sample, and evaluate each by RT-PCR. This work will establish a reliable protocol for sorting spermatids and will yield ordered libraries of most or all of the active and stored spermiogenic information. Our long-term goals are to use this technology to catalog and characterize most or all spermiogenic mRNAs and to determine when each one is used during sperm maturation. In the current submission, the applicant is requesting two years of R03 funding to rigorously establish the technology on which this research, as well as countless other molecular investigations on spermiogenesis, can be founded.

描述（改编自申请人的描述）：精子发生是 基于精心定时的存储翻译的顺序组装过程 转录沉默细胞中的 mRNA；然而，分子机制 决定单个 mRNA 何时被翻译的机制尚不明确 明白了。很大一部分问题是由于我们没有可靠的 纯化不同阶段精子细胞亚群的方法 分子分析的成熟。拟议的研究旨在解决这个问题 不足。 申请人提议开发一种使用转基因的方法 表达水母绿色荧光蛋白（GFP）的小鼠 减数分裂雄性生殖细胞与荧光激活细胞结合 分选（FACS），以纯化阶段精子细胞的同质亚群。 该项目的目标是： 1) 分离至少 20 个精子细胞子 通过 FACS 进行人群。 2）评估精子细胞的纯度和阶段 基于形态学、超微结构、 和分子标准；选择 10 个同质子群体代表 跨越所有精子发生的不同阶段。 3) 纯化mRNP颗粒- 相关 mRNA（存储信息）和多核糖体相关 mRNA（活性 信息）从每个同质亚群中，准备一个 cDNA 从每个 mRNA 样本中提取文库，并通过 RT-PCR 进行评估。这项工作将 建立可靠的精子细胞分选方案并产生有序的 大部分或全部活跃和存储的精子发生信息的文库。 我们的长期目标是使用这项技术来编目和表征 大多数或所有生精 mRNA 并确定每个生精 mRNA 的使用时间 精子成熟。 在当前提交的材料中，申请人请求两项 多年的 R03 资助严格建立了该技术的基础 研究以及无数其他关于精子发生的分子研究， 可以成立。